The mammalian cell cycle is regulated by a complex network of temporally oscillating regulatory proteins that either promote or halt cell growth and division. Misregulated cell cycle control is a fundamental aspect of cancer. The SCF E3 ubiquitin ligase is a dynamic protein complex that manages the cell cycle by targeting the oscillating regulatory proteins for ubiquitin-dependent degradation. Misregulation of several SCF ligases that control cell cycle progression is implicated in numerous malignancies. While the misregulation of additional SCF ligases may also be involved, the complete repertoire of SCF ligases is yet to be characterized. Therefore, a thorough understanding of how SCF ligases regulate the cell cycle is critical for identifying optimal therapeutic targets. The SCF ligase complex contains 3 invariant subunits (Skp1, Cul1, and Rbx1) and 1 invariant substrate recognition subunit (F-box protein (FBP)). The human genome encodes for 69 FBPs, which are the SCF substrate recognition subunits, suggesting that cells may assemble up to 69 distinct SCF complexes. Comprehensive analysis of which FBPs assemble into an SCF complex and how the complex reorganizes in response to cell cycle progression is lacking. My preliminary data identified numerous uncharacterized FBPs that are expressed constantly during the cell cycle but assemble into SCF ligases in a cell cycle dependent manner. This suggests that these ligases function to regulate the cell cycle and that FBP assembly is not coordinated by FBP expression but rather by cell cycle phase-dependent substrate availability. Therefore, the objective of this proposal is to determine the temporal dynamics of SCF E3 ubiquitin ligase assembly throughout the cell cycle and to then study the unique biological functions of SCF ligase and the mechanisms regulating complex assembly. This objective will be tested through three specific aims. Aim 1 will identify the repertoire of SCF E3 ubiquitin ligases that temporally assemble in different cell cycle phases. This aim will be addressed by utilizing a multi-modal approach combining multiplex-selected reaction monitoring (SRM) mass spectrometry and flow cytometry to quantify the abundance of FBPs, invariant SCF subunits, and regulatory proteins bound to Cul1 versus unbound in different cell cycle phases. Aim 2 seeks (1) to determine how SCF ligases that assemble in a cell cycle dependent manner regulate cell cycle progression by evaluating cell cycle progression in cells depleted of oscillating FBPs and (2) to use mass spectrometry to identify substrates for those FBPs that influence the cell cycle. Aim 3 seeks to determine if substrate availability drives the temporal assembly of FBPs into SCF complexes during the cell cycle. This aim will be tested by manipulating FBP-substrate interactions and measuring the impact on assembly of the cognate SCF. The overall results of this study will not only improve our understanding of cellular proliferation and SCF biology but also provide a foundation for understanding how misregulation of FBPs contributes to tumorigenesis.